Science and theory. (Tools for Environmental Health).

When the first news of anthrax-containing letters hit the airwaves and the print media, there came a spate of "white powder" panic calls to many police and health departments across this nation. For about a month or two thereafter, first responders were sent scurrying to collect the offending parcels and envelopes and to remove dust, including powdered sugar from doughnuts that would surely cause widespread infirmity and death. A frightened public demanded assurances that public buildings--particularly schools--were safe.

We fielded what seemed to be an endless stream of calls from businesses, schools, governmental agencies, and private citizens asking for our expertise in sampling and testing of the environment for the deadly anthrax bacillus. Although our backgrounds in institutional practice prepared us with some degree of comfort, it soon became apparent that, collectively public-health officials did not have a good grasp of aerobiology or the science of monitoring air for biological agents other than molds. Not surprisingly, we turned to the familiar and hoped for the best. For instance, we relied on sampling techniques used in industrial hygiene rather than those used by the biomedical and pharmaceutical industry for validation of client plant and processes, and we manipulated each sample as if it were an enriched clinical specimen rather than one coming from a stressed environment. Not that this approach is entirely wrong, but using it makes us much like a reverse soothsayer who divines the question after being given th e answer.

For this reason, we are departing a bit from our regular format and presenting Part One of a three-part primer on environmental air and surface sampling. We will try to present this technology in a logical format--since we had to do that for our clients. Our goal is to provide some basic information from which to ask appropriate questions. This first segment will focus largely on the science and theory, the next on instrumentation and technique. The final segment will address surface sampling and introduce a few new and innovative methodologies.

The Science

There are four cardinal premises in environmental microbiology. Understanding the rationale of these premises will have a significant and positive impact on our choice of sampling techniques, our approaches to analysis, and, ultimately, accuracy in our interpretation of data.

The first, and probably most important, premise: Most microbes do not survive well outside of their natural environment or growth site. For instance, a human pathogen that thrives in the gut, where it has an enriched, warm, moist environment, does not do well on a dry, cool surface or in low-humidity air. Organisms found in the latter types of environments are usually stressed, We therefore expect to find a greater number of microorganisms associated with humans in a room with a lot of activity and considerably fewer--and different types--of organisms in a room that has been vacant for some time.

Premise 2: Microbes are found everywhere, in virtually every environment, both natural and man-made. Therefore, when sampling, expect to find nontarget organisms that may be far more robust than those you are looking for. Expect the unexpected.

Premise 3: There is no uniformity of distribution. Different environments differ with respect to quality and quantity of microbes. The greater the proximity to an aerosolizing source or portal of exit (if you are dealing with the chain of infection), the greater will be the number of microorganisms. The same holds true for growth conditions. More microbes will be found where conditions favor their growth and reproduction. Because microorganisms have mass, they behave as particulates. More organisms will be on horizontal than on vertical surfaces, and more at the floor than at the ceiling.

Finally, Premise 4: Each environment can be considered a separate biosphere, each with a characteristic bioburden. Therefore, we need to estimate the bioload, both qualitatively and quantitatively, for each environment or portion thereof we want to sample or evaluate. The estimate will guide us to the appropriate collection and analytical methodology, and serve as a template for interpretation of the data.

Sampling Theory

Sampling strategies differ for regulatory-compliance and forensics applications. Each application requires a different degree of sampling sophistication and a different approach to data analysis. The instrumentation, in this case microbial aerosol samplers, must be judged in terms of its capability to collect microbes under different operating conditions while minimizing the environmental stress on the organisms collected. Therefore, no single sampling method is suitable to all occasions.

Another point to remember: Rarely is microbial air sampling conducted as a quality control point. More often than not, air and surface sampling is done to determine the cleanliness, or lack thereof, in an environment of public-health significance. Or, in the anthrax case, the purpose is to answer the question: How clean is clean?

Microbial air samplers are characterized by mode of capture, flow rate and flow characteristics, and collection efficiency as a function of particle size and shape. As a rule, aerosol collection devices that exhibit the lowest shear forces collect samples in which microorganisms have the highest viability. Conversely, these samplers usually have the lowest physical efficiencies in terms of numbers of airborne particles collected. Therefore, the efficiency of microbiological collection depends largely on the sampling method used.

The primary objective of any sampling program is to produce a set of samples that are representative of the source under investigation and that are suitable for subsequent analysis. Because the air is not homogeneous in any environment, there can be no duplicate samples. We therefore need to consider sampling conditions, sampling time, and sample size as limitations in our data collection scheme.

Collecting a sample of airborne microbes that is representative is probably the most difficult goal to achieve. Apart from the inherent absence of microbial uniformity in air, there is the problem of ensuring that particles of all sizes have an equal probability of entering the sampler. This problem can be partially addressed when the sampling rate is chosen so that the velocity of air entering the sampler inlet equals the velocity of the air being sampled. Achieving or approaching isokinetic sampling conditions is particularly important in an area in which air sampling is done under dynamic conditions. If the velocity of the air entering the sampler is greater than that of the room's air movement, small particles will predominate because they move more easily across the streamlines. Conversely, if the velocity is slower, larger particles will predominate because, unlike smaller particles, they do not follow the curvature of the streamlines around the sampler inlet. Anisokinetic sampling may result in samplin g errors that range from 20 percent to 300 percent, depending on particle size and environmental conditions. Ideally, to avoid the effect of dynamic air movement on the capture of microbial aerosols, stagnation point sampling would be used. In the absence of any air movement, smaller particles are efficiently captured and a particle-size profile estimated.

To further complicate this issue, consider the following: Air is not a natural environment for most microbes. Survival of microorganisms in air is affected by a large number of environmental factors, the most important of which are temperature and humidity. Under natural conditions, these numerous factors operate simultaneously. Consider also that force is required to generate an aerosol and, likewise, to capture particles within that aerosol. These forces can damage or even fracture fragile structures such as microbes in their vegetative state. The fragile nature of airborne microorganisms is largely species dependent and is determined by physiological condition. Once airborne, microbes become stressed through desiccation or hydration, depending upon the condition of their natural growth site. Radiation, oxygen, ozone, and various other gaseous and particulate pollutants, if not lethal, may further stress the organisms. Some stressed and injured microorganisms may, however, fully recover when given a suitabl e environment. This property of reversible injury or repair in microorganisms is widespread, and the implications of it are important in development of the testing protocol.

Analytical Considerations

The agar medium selected for use in all microbiological sampling devices should be fresh, and you should prescreen it for sterility by placing it in an incubator at 36[degrees]C for 24 hours. In the initial microbial assays, malt extract agar is recommended for the general detection of fungi, while agar containing casein peptone, soy peptone, and sodium chloride is used for bacterial sampling. Trypticase soy agar has probably the most universal applicability for the collection of aerobic bacteria and fungal species, whereas, for the detection of anaerobic species, a thioglycollate medium is recommended. Once initial bioburden estimates are established and a greater specificity is required to target certain organisms, specialized agars containing antibiotics and/or other inhibitory and growth-regulating compounds are available from commercial sources. Use of these selective media in an air-sampling device, however, may severely hinder collection efficiency These media are generally inhibitory to small inocula, even of the organisms for which they are "selective," by retarding recovery of those that are injured or stressed.

Because the organisms found in air come from different environments, the temperatures used to enhance their growth on an artificial medium should approach that of their normal habitat. Most of the organisms found in the air fall within two distinct temperature preferences. The psychrophilic organisms, or those that prefer cold, thrive at low temperatures between 0[degrees] and 30[degrees]C, while the mesophilic organisms prefer moderate temperatures between 15[degrees] and 43[degrees]C. Therefore, to recover the maximum number of organisms in any air sample, consider incubating the medium at 22[degrees]C for 24 hours (or 48 or 72 hours, as necessary) to recover psychrophilic organisms, immediately followed by incubation at 36[degrees]C to recover the mesophilic organisms.

The outcome of a well-planned sampling strategy depends on good science, logic, and, to a lesser degree, a measure of good luck. Taking the time to estimate the types of organisms that may be present; describing the static, dynamic, and physical characteristics of the area under test and its air; and conducting a viable/nonviable-particulate profile of the space to be sampled will yield data that become the basis for the entire microbiological sampling scheme.

There is no single agar medium on which all microorganisms will grow no single incubation temperature that will encourage growth, and no single assay procedure that can completely characterize the microbial contamination in all environments. Likewise, there is no universal sampling device.

Next month we will review the types and uses of biological air samplers.

Inspection Tip of the Month

When inspecting retail food establishments, take with you at least half a dozen inexpensive calibrated bimetal thermometers. When you first enter the premises, place these thermometers in the various hot- and cold-holding facilities. Doing so will save considerable time in monitoring equipment temperatures, as well as minimize any potential stochastic errors in measurements.

This article is provided by NEHA for informational purposes only. It is designed to better inform our members about topical and relevant goods and services available to the environmental health professional. Opinions expressed about any product or service in this column either expressly or implied are solely and completely those of the author/s and do not necessarily represent the views or opinions of the National Environmental Health Association.